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The toxicological effects of chlorinated paraffins in mammals
TOXICOLOGY
AND
APPLIED
PHARMACOLOGY
The Toxicological ROBIN DUNCAN
Imperial
Effects
54,
514-525 (1980)
of Chlorinated
Paraffins
in Mammals
NICHOLAS BIRTLEY, DAVID MICHAEL CONNING,’ JOHN WYNFORD DAVID MACKAY FERGUSON,~ ERIC LONGSTAFF, AND ANDREW ALEXANDER BUCHANAN SWAN
Chemical
Industries
Limited, Central Toxicology Laboratory, Cheshire SK10 4TJ, England
Received
September
4, 1979; accepted
March
Alderley
Park,
DANIEL,~
Macclesjield,
28, 1980
The Toxicology of Chlorinated Paraffins in Mammals. BIRTLEY, R. D. N., CONNING, D. M., J. W., FERGUSON, D. M., LONGSTAFF, E., AND SWAN, A. A. B. (1980). Toxicol. Appl. Pharmacol. 54, 514-525. Chlorinated paraffins are a group of chemicals derived from n-paraffins containing between 10 and 30 carbon atoms which are chlorinated to between 40 and 70% (w/w). Representative grades were tested for acute oral toxicity and skin irritancy in rats, eye irritancy in rabbits, and potential mutagenic effects in two in vitro tests. The effects of one grade (14-17 carbon atoms, 52% chlorinated) which is manufactured in high-tonnage quantities, was evaluated in a 90-day feeding study in rats and dogs, and its tissue retention and elimination was determined in the rat. Chlorinated paraffins are of low acute oral toxicity. Slight, reversible skin and eye irritations were produced by shorter carbon chain derivatives only and no type showed any evidence of mutagenic potential. Subacute administration of 500, 2500, and 5000 ppm reduced diet palatability to rats. Increases in SGPT or SAP levels, liver weight, and smooth endoplasmic reticulum in hepatocytes were seen in rats and dogs: “no-effect” diet levels were 250 and 300 ppm, respectively. Chlorinated paraffin accumulates primarily in the abdominal fat of rats at concentrations which never exceed those in diet (unlike certain other chlorinated organic compounds); its half-life in abdominal fat is about 8 weeks, when untreated diet is offered. On the currently available evidence, in view of their slight toxicity and the low human dietary levels in comparison to the maximum “no-effect” levels in rats and dogs, it is concluded that chlorinated paraffins present no toxicological risk to man despite their continued manufacture and widespread low-level occurrence in the environment. DANIEL,
Chlorinated paraffins are a large group of industrially important chemical types, the extremes of which are n-paraffins of 10 and 30 carbon atoms chlorinated to 40 and 70% by weight. There are many grades of chlorinated paraffin, each of which is a complex mixture of compounds and iso’ Present address: British Industrial Biological Research Association, Woodmansteme Road, Carshalton, Surrey SM5 4DS, England. * Present address: Life Science Research, Stock, Essex CM4 9PE, England. 3 Present address: Imperial Chemical Industries Americas Inc., Wilmington, Del. 19897. 0041-008x/80/090514-12$02.00/0 Copyright All rights
D 1980 by Academic Press, Inc. of reproduction in any form reserved.
mers representing a small spectrum within the ranges of carbon chain length and extent of chlorination. Each grade has specific physical and chemical properties which render them useful in such widespread applications as plasticisers in plastics such as polyvinyl chloride, extreme pressure additives for gear and cutting oils, fire-retardant additives, paints, and electrical insulators. They have been in use for up to 40 years. There are no reports in the literature of the toxicological effects of chlorinated paraffins in mammals. The studies reported here describe the results obtained in two in 514
TOXICOLOGY
OF CHLORINATED
tests which predict mutagenic potential and in mammalian acute toxicity and irritancy tests with a variety of chlorinated paraffin grades which represent the spectrum of chemical types and which are manufactured in significant quantities. Also, results of subacute toxicity studies in rats and dogs together with tissue retention and elimination studies in rats are described for one grade of chlorinated paraffin which is manufactured in large quantities. These are the studies which have been completed to date in a major ongoing program designed to evaluate the toxicological potential of chlorinated paraffins.
vitro
METHODS 1. Salmonella typhimurium (Ames test)
Reverse Mutation
Assay
The following chlorinated paraffin grades were evaluated in this test: Cereclor’ S52 (14- to 17-carbon n-paraffins, chlorinated to 52% on average), this grade was evaluated without and with 0.2% (w/v) epoxidized vegetable oil added as stabilizer; Cereclor’ 42 (20- to 30-carbon n-paraffins, chlorinated to 42% on average); Cereclor’ 50LV (lO- to 13-carbon n-paratlins, chlorinated to 50% on average). The method used was essentially the same as that described by Ames et al. (1975). Each grade was tested at five dose levels (2500, 500, 100, 20, and 4 pg per plate) on each of 5 consecutive days, and each dose level was tested in duplicate. Four strains ofSalmonella typhimurium were used throughout, namely TA1535, TA1538, TAlOO, and TA98, both with and without Aroclor-induced rat liver postmitochondrial supernatant (S-9 mix). 1,3-Propanesultone was used as positive control substance for TA1535 and TAlOO strains while 2-acetylaminofluorene was used for the other two strains. Dimethyl sulfoxide was used as vehicle for all test substances and as the negative control substance.
’ The name “Cereclor” is a trademark, the property of Imperial Chemical Industries Limited, for the chlorinated paraffin product range. The letters and digits identify further the specific grades. All chlorinated paraffins were supplied by ICI Mond Division, PO Box 8, The Heath, Runcorn, Cheshire WA7 4QD, England.
PARAFFINS
2. Cell Transformation
515 Test
The same three grades of chlorinated paraffin which were evaluated in the S. typhimurium test were also tested here. The procedure was identical to that described by Styles (1977). Five concentrations of each test compound in the final mixture were used, namely 2500,250, 25,2.5, and 0.25 &ml. 2-Acetylaminofluotene (1250 &ml) was the positive control substance. 3. Acute Toxicity and Irritancy Studies (a) Acute oral toxicity to rats. A single oral dose of a chlorinated paraffin, either as the material as supplied or in a vehicle such as olive oil, was administered to groups of at least three female and/or male Alderley Park Wistar rats.* In any one experiment, the body weights of all animals were within 50 g, but the weights of the animals for all studies were in the range of 125-250 g. They were deprived of food for 16 hr prior to dosing, and then allowed food and water ad [ibitum during the remainder of the experiment. The dose levels used were between 0.5 and 10 g/kg body weight. The animals were regularly observed for abnormal clinical signs for up to 14 days after treatment and an autopsy was performed at the end of the observation period (or at death if this was sooner). Occasionally, certain organs or tissues which were considered to have been affected by treatment were processed by standard histological techniques and sections were examined microscopically. (b) Skin irritancy evaluation. A total of up to six applications of each grade tested was made to the shorn back of each of three female rats* (125-250 g body wt) on alternate 24-hr periods. The volume applied was generally 0.1 ml, either as the neat chlorinated pa&tin or as a concentrated solution in a nonirritant vehicle. The treated skin was kept covered with an occlusive dressing while in contact with the test agents, and at the end of each 24-hr contact period the skin was washed once with a weak detergent solution. The rats were housed singly during the experimental period and allowed free access to food and water. Skin reaction to treatment was subjectively assessed 24 hr after removing each application and on some occasions immediately after washing the skin. The severity of erythema, desquamation, peeling, necrosis, etc., were recorded for each rat on all occasions. If severe reactions occurred in any animal before the scheduled experimental period ended, the testing in that animal was terminated prematurely. In certain instances such animals and some which completed the * Supplied by the Animal Breeding Unit, Imperial Chemical Industries Ltd, Alderley Park, Macclesfield, Cheshire, England.
516
BIRTLEY
ET AL.
TABLE NUMBER
OF SAMPLES
1
OF CHLORINATED
THREE
TYPES
PARAFFINS
OF ACUTE
EVALUATED
TOXICITY
IN EACH
OF
TEST
Extent of chlorination
of n-paraffin (by weight) 51-60%
41-50%
61-70%
Number of carbon atoms in n-pa&in chain
AOb
SIC
EI”
A0
SI
EI
A0
SI
El
10-13
3
8
0
0
0
6 9
0 6
9 0
3
0
2 8
11
14-17
20-30
2
1
0
1
1
0
2
00 2
2
a AO, Acute oral toxicity. b SI, Skin irtitancy evaluation. c EI, Eye irritancy evaluation. d Each sample of chlorinated paraffin is derived from a mixture of n-paraffins containing the range of numbers of carbon atoms shown, but it is chlorinated to a specific percentage by weight. The table shows the number of samples evaluated in each type of test whose percentage chlorination falls within the arbitrary ranges shown. duration of the experiment were subjected to autopsy procedures and any abnormalities were noted. (c) Eye irritancy evaluarion. A single application (usually 0.1 ml) of undiluted chlorinated paraffin was instilled into the conjunctival sac of one eye of each of three New Zealand White rabbit? (2.5-3.5 kg body wt) of either sex. The other eye served as a contralateral control. The rabbits were housed singly and allowed food and water ad l&turn during the experimental period. The irritancy induced was scored 1 hr after instillation then at daily intervals up to 7 days afterward, by the method of Draize (1959) as modified by Kay and CaIlandra (1962). A subjective assessment of initial pain reaction was made in some experiments. Any animal seen to be unduly stressed as a result of the treatment was killed. The number of grades of chlorinated paraffin, classified broadly according to the number of carbon atoms in the n-paraffin chain and the extent of chlorination by weight, which were evaluated in acute toxicity and irritancy tests are summarized in Table 1. Identical grades, which contain small amounts of different types of stabilizer, have been counted as separate test compounds.
4. Subacute
Toxicity
(a) Ninety-day feeding containing stabilizer.
Studies study
in rats with Cereclor
S52-
Specific pathogen-free, Wistarderived rats (body weight, 160- 180 g) were randomly selected from a larger population* and were housed in wire mesh cages suspended on racks. Groups of
48 rats (24 males, 24 females) were housed four per cage and maintained for 90 days on diets containing 500, 2500, or 5000 ppm Cereclor ,852~containing stabilizer; the diet of a similar control group of 48 rats contained the vehicle, corn oil only. Food3 and water were available ad libitum. At 6 weeks, a further group of 48 rats whose diet contained 250 ppm Cereclor S52 with stabilizer was introduced into the experiment together with an additional control group of 12 male and 12 female rats; these rats were treated in an identical manner to those on the main study. The concentration and distribution of the chlorinated paraffin in each batch of diet were checked by chemical analysis. Each rat was observed daily throughout the treatment period for clinical abnormalities. The body weight of each rat was recorded initially and at weekly intervals together with the food consumption per cage. Samples of blood were taken from 10 males and 10 females from each group preexperimentally, at 6 weeks and terminally for individual determination of haemoglobin concentration, packed cell volume, mean corpuscular diameter. reticulocyte count, total and differential white cell counts and platelet counts. Clotting function tests (prothrombin index and kaolincephalin time) were performed terminally. Plasma alkaline phosphatase and glutamic pyruvate transaminase activities were assayed in samples from four males and four females from each treatment group preexperimentally, after 6 weeks and terminally. At the end of the 90-day period all animals were killed with a dose of volatile anaesthetic and an im3 Oakes Ltd., Congleton, Cheshire,
England.
TOXICOLOGY
OF CHLORINATED
mediate full postmortem examination made. The liver, kidneys, adrenals, lung, heart, and spleen from half of the animals of each sex in each group were weighed and organ-to-body weight ratios calculated. The following organs were examined microscopically after preservation in form01 corrosive, processing to paraffin wax blocks and hematoxylin and eosinstained sections (5 pm) mounted on slides: liver, kidney, spleen, heart, lung, adrenal, gonads, thymus, thyroid, pancreas, stomach, duodenum, jejunum, cecum, ileum, colon, salivary gland, and mesenteric lymph nodes. The brain (cerebrum, cerebellum, and pans) and spinal cord were fixed in 10% form01 saline prior to processing and sectioning. Sections of liver from three male and three female animals from each group, which had been deprived of food overnight, were preserved for electron microscopic examination. Sections of liver, kidney, spleen, adrenals, pancreas, lung, gonad, stomach, duodenum, ileum, and colon from similar groups of animals were preserved in liquid nitrogen for histochemical analysis of certain enzymes. fh) Ninety-day feeding study containing stabilizer. Beagle
in dogs with Cereclor
S52
dogs from an inbred strain’ (body weight, lo-14 kg), which had been immunized against distemper and hardpad were housed in individual pens and, prior to experimental assignment, received two doses of piperazine adipate5 to exclude nematode infection. Their main meal, offered at noon each day, comprised 45 g of a meat preparation” and 226 g of a dried pelleted diet:7 a dog biscuits weighing 56 g was offered each afternoon. Each animal had its own automatic drinking fountain. Cereclor SS2-containing stabilizer was diluted with corn oil and mixed in a ball mill for 24 hr. The concentration was adjusted to allow dosage at the rate of 1 ml/kg body wt per day, and the resultant solution was syringed into and thoroughly mixed with the main meal. Four male and four female beagles at each dose level were dosed daily for 90 days with 0, 10, 30. or 100 mgikg body wt of Cereclor S52. The distribution and final concentration of chlorinated paraffin in diets were checked by chemical analysis. Each animal was observed daily for gross clinical effects; detailed examinations were made preexperimentally and terminally. The body weight of each dog was determined preexperimentally and then weekly throughout the experiment. Samples of blood were taken from each dog pre’ Epivax, Burroughs Wellcome and Co., London. j Coopane, Cooper, McDougall and Robertson Ltd., Berkhamsted, Hertfordshire. England. ’ Kennomeat, Scottish Animal Products, Barrhead, Scotland. ’ Kennel Kernals. James and Co.. Hungerford, Berkshire, England. ’ Winalot, Spillers Ltd., London, England.
517
PARAFFINS
experimentally and after 42 and 90 days of treatment for determination of hemoglobin, packed cell volume, mean cell hemoglobin concentration, mean cell diameter, reticulocyte count, leukocyte count (total and differential), platelet count and clotting function (kaolin-cephalin time and prothrombin index). Bone marrow cytology was investigated by needle biopsy from the iliac crest between Days 40 and 50 and terminally. Also, for all dogs in each group, blood glucose, blood urea, plasma alkaline phosphatase, glutamic pyruvic transaminase, sodium, and potassium were determined at the same times as the hematology parameters were measured. Urinalysis (pH, specific gravity, protein, glucose, and bilirubin) was undertaken in samples of urine obtained overnight from each dog preexperimentally and terminally. Electrocardiography was also performed preexperimentally and terminally. At the end of the 90-day feeding period all dogs were killed by intravenous administration of a lethal dose of pentabarbital sodium and a full postmortem examination was made. Heart, liver, kidneys, adrenals. spleen, thyroids, testes, lungs, and brain were weighed and organ-to-body weight ratios calculated. Representative pieces of tissue were taken from the pituitary, salivary gland, thyroid, thymus, heart, lung, aorta, stomach, duodenum, jejunum, ileum, colon, liver, spleen, kidney, bladder, adrenal, lymph nodes, ovary and uterus, testis, and epididymis; fixed, processed into paraffin wax, 5-pm sections stained with hematoxylin and eosin and examined microscopically. Samples of brain, spinal cord and sciatic nerve were taken from all groups. although only those from the controls and highest dose group were processed and examined. Samples were also taken from the major lobes of the livers of all animals and processed for examination in the electron microscope. Similar samples were frozen in liquid nitrogen and processed for a range of enzyme histochemical reactions: in addition the sections were analyzed for the numbers of nuclei per unit area as a measure of cell size. 5. Tissue Cereclor
Retention and S52 in Rats
Elimination
of Radioactive
lS”Cl]Cereclor S52 was synthesized by chlorination to 52% (w/w) of commercial C,,-C,, n-paraffin feedstock with [3”Cl]chlorine which had been generated from a solution of sodium [36Cl]chloride (41.7 &i/ml)Y by reaction with hydrogen peroxide and oleum; the specific activity of the purified product was 0.36 mCi/g. Male Wistar rats (180-200 g body wt) of the Alderley Park Strain2 were used throughout. Food3 and y Radiochemical shire, England.
Centre, Amersham, Buckingham-
518
BIRTLEY
ET AL.
TABLE THE MEAN DAILY NUMBER OF CHLORINATED PARAFFIN, FOUR STRAINS OF Salmonella
2
OF REVERTANT COLONIES PER PLATE FOR EACH DOSE TESTED OF THREE GRADES Two POSITIVE CONTROL SUBSTANCES, AND A VEHICLE CONTROL WITH EACH OF typhimurium IN THE PRESENCE AND ABSENCE OF RAT LIVER S9-MIX
Strain of S. typhimurium TA1535 Test substance
Dose ( wdplate)
+
without (-) or with (+) SPmix
TA1538 -
TAlOO
TA98
+
-
+
-
+
-
Cereclor S52 without added stabilizer
2500 500 100 20 4
11 18 29 16 25
9 9 8 8 8
22 32 12 58 63
6 7 4 6 7
53 46 43 42 34
48 42 45 46 39
12 15 15 18 14
14 9 9 11 10
Cereclor S52 with added stabilizer
2500 500 100 20 4
16 24 18 15 21
13 9 8 9 10
24 10 11 14 13
8 9 8 10 10
50 37 42 41 42
31 30 25 35 33
20 13 11 13 8
10 10 12 9 11
Cereclor 42
2500 500 100 20 4
12 14 14 12 13
8 7 8 9 7
14 12 15 14 15
8 6 8 9 6
36 35 30 39 33
35 26 32 33 30
19 14 12 15 11
7 10 9 6 8
Cereclor 50LV
2500 500 100 20 4
10 6 11 8 6
7 7 11 8 8
8 11 14 15 8
10 7 12 12 7
37 38 33 26 37
34 29 26 24 28
11 11 9 14 10
7 7 7 6 7
1,3-Propane sultone
500 100 20
489 287 148
340 219 161
2-Acetylamino fluorene
500 100 20
213 251 208
9 10 13
Dimethylsulfoxide (vehicle control)
-
30
10
water were available ad libirum mental periods.
38
during the experi-
(a) Tissue retention and elimination studies. The retention of radioactivity was investigated in two groups of rats which were maintained on diets containing either 0.4 or 40 ppm [30Cl]Cereclor S52. The required amount of Cereclor S52 was dissolved in corn oil which was then added to malt extract (18 parts) and water (5 parts) and the whole mixed mechanically with stock diet3 (77 parts). Animals were fed the experimental diet ad libitum for either 8 (40 ppm) or 10 weeks (0.4 ppm). Groups of three animals were killed
11
546 346 277 280 188 198 165 148 118
8 9 8
20
10
74
60
at weekly intervals. After 10 weeks, the surviving animals which had been maintained on the 0.4-ppm diet were returned to a control diet and three rats were killed at varying times during the subsequent 8 weeks. Abdominal fat, liver, brain, and adrenal glands were removed from all rats immediately after death and the quantity of radioactivity in them was measured. (b) Analytical
methods for measurement
of radioactivity
Abdominal fat and brain were macerated in p-dioxane (20 ml) and liver was homogenized in acetone (20 ml). The suspensions were centrifuged and the supernatant solutions retained. The radioactivity in these
TOXICOLOGY
OF CHLORINATED
PARAFFINS
519
fluorene caused greater than five times the background transformation frequency of approximately 50 per lo6 surviving cells, showing that the test was working satisfactorily. None of the concentrations of the chlorinated paraffins tested caused the cell transformation frequency to exceed background values, even at the LCsO value. RESULTS The relative lethality of the four test subI. Salmonella typhimurium Reverse Mutation stances to baby hamster kidney cells is of Assay (Ames Test) interest. The LC,,, values for Cereclor SOLV, 42, S52 with stabilizer, and S52 without stabiThe results are summarized in Table 2. lizer were estimated from dose-lethality There was no indication that chlorinated curves to be approximately 1.3,36,228, and paraffins were toxic to any of the four tester 1250 pg/ml, respectively. strains of S. typhimurium at doses up to 2500 pg per plate. The test system was shown to 3. Acute Toxicity and Irritancy Tests be working satisfactorily as dose-response relationships were established for 1,3-pro(a) Acute oral toxicity. Out of all the difpanesultone, and activation of 2-acetylferent grades of chlorinated paraffins tested aminofluorene in the presence of S9-mix (Table l), clinical signs of toxicity were conwas evident; both positive control sub- fined mainly to rats which received a high stances displayed unequivocal mutagenic dose (4 g/kg body wt or above) of chloriproperties in this test. Using the criterion nated paraffins with lo-13 carbon atoms that the ratio of the test to negative control in the n-paraffin chain. Observations of response should be at least 2.0 to provide toxicity which were commonly made were evidence of a mutation, it can be seen from nonspecific and included piloerection, Table 2 that no such evidence exists for muscular incoordination and urinary and any of the chlorinated paraffin grades fecal incontinence. These animals usually tested. The test response was greater than recovered completely within 7 days of treatdouble the negative control value when the ment. The intensity and nature of these two lowest doses of Cereclor S52 without responses was generally independent of the added stabilizer were evaluated with the chlorine content of the substances tested. TA1538 strain in the presence of S9-mix. Thus the acute oral LD,, of all grades This is considered to be an anomalous find- tested exceeded 4 g/kg and in some cases ing since, in the absence of evidence for a 10 g/kg. direct toxic action of the chlorinated parThe few abnormalities observed at necaffin on the cells, a dose-response rela- ropsy and on microscopic examination of tionship would be expected if a real mutatissue sections were generally confined to genic effect was being exerted. Furtherthose rats which showed signs of toxicity. more, no indication of a mutagenic effect Occasional inflammation of the gastric muwas seen in any strain treated with Cereclor cosa was observed, indicating a localized S52 which contained stabilizer. irritant action following administration of some chlorinated paraffins. Histopathological abnormalities were confined to the 2. Cell Transformation Test liver (hepatocellular vacuolation and ocThe results are presented graphically in casional necrotic foci) and the kidney (cloudy swelling of some inner cortical cells). Fig. 1. In each experiment, 2-acetylaminoextracts was measured using an Intertechnique SL 30 liquid scintillation spectrometer as described by Daniel and Gage (1965). The adrenals and samples of airdried solvent-insoluble material from other tissues were combusted in an atmosphere of oxygen, and the [36Cl]chlorine absorbed and counted using a modification of the method of Dobbs (1963).
520
BIRTLEY
ET AL.
Ifqy %surYl”ors I
1.100
iI
1.100
: :
900
:
I : :
900.
: I
: 700
I
700
I
500.
i :
300.
: I I
:
Wansformants per 10’ survivors
:
500
tronsformonts per lo* survivors
:
I 100 ! .----------__--__________ .----------____________,
looI 0
0.25
-------------,--‘,,,,,,,------
100 ’ 2.5
25
Concentmtion
250
2% lo
0
“g/ml
0.25
2.5
25
Conwntmtion
4 2500
250
us/ml
I. 1.100 I
i I
I transformants per 106 survivors
500
i tranrformants par lo’ survivors
:
I
0
i
0.25
1.5
Concsntmtion
25
250
lo
ug/ml
500
0
0.25
2.5
Concontrotion
25
250
2!
0
ug/ml
FIG. 1. Cell transformation test. Survival and transformation frequency of baby hamster kidney cells exposed to various concentrations of Cereclor 50LV (top left), Cereclor 42 (top right) Cereclor S52 with added stabilizer (bottom left), and Cereclor S52 without added stabilizer (bottom right). The transformation frequency is measured at the LC,,; it should exceed 2.50 per 106 survivors (five times the background frequency) for a potential carcinogenic effect to be established.
(b) Skin irritancy evaluation. Under the stringent conditions of this test, most chlorinated paraffins showed some degree of skin irritation. A mild skin irritancy response was confined to some of the grades derived from n-paraffins containing lo- 13 and 14- 17 carbon atoms; the response was generally independent of the degree of chlori-
nation. The occasional responses which were of moderate intensity were produced by the C,,-,, n-paraffins chlorinated to 70%. The mild. erythema and desquamation responses were usually established by the third application, but subsequently the condition did not worsen and often improved. It is possible that the irritancy response
TOXICOLOGY
OF CHLORINATED
was partly due in some cases to the presence of stabilizer in the test substance. All the longer carbon chain chlorinated paraffins were nonirritant. In experiments where autopsy was performed at the end of the treatment period, no evidence of systemic toxicity could be found. (c) Eye irritancy evaluation. Of the few grades evaluated in this test only mild, transient eye irritation was seen, and this was confined to the short carbon chain n-paraffins. Specifically, slight redness of the conjunctiva lasted no more than l-2 days; no abnormalities were noted in any other ocular structures and no pain response was observed in any animal.
PARAFFINS
521
limited quantities of the diet containing 500 ppm Cereclor S52. Over a period of 28 days the food conversion ratios of these two groups were comparable to that of a group fed control diet ad libitum indicating that the reduction in body weight gain in the main experiment was related to reduced palatability of the diets containing up to 2500 ppm Cereclor S52. From the increased food conversion ratio seen in male rats receiving 5000 ppm in diet, an adverse effect on weight gain is also suspected (Table 3). The results of the haematological investigation showed no abnormality attributable to the test compound, although male rats which received 5000 ppm Cereclor S52 showed a reduction in kaolin-cephalin index which was not statistically significant. A marked decline in alkaline phospha4. Subacute Toxicity Studies tase activity over the test period was seen in all groups, including controls. The se(a) Ninety-day feeding study in rats with rum glutamic pyruvate transaminase acCereclor S52. Diet analysis showed adequate tivity also showed a tendency to decline mixing of chlorinated paraffin with the except in male rats receiving the highest food and the achieved concentrations were dose where it increased somewhat. similar to theoretical values. There were At gross postmortem examination the no deaths during the course of the experionly specific abnormality revealed was a ment and no clinical abnormalities; the be- tendency towards congestion of the kidney haviour of all groups appeared normal. with increasing dietary concentration of The group mean body weight of treated Cereclor S52. Microscopically there were male rats was significantly lower than no abnormalities that could be attributed that of the control rats from Week 4 to the test compound. No abnormalities in onward at 500 ppm (p < 0.05) and from organ-to-body weight ratio were observed Week 1 onward at 2500 and 5000 ppm (p in animals receiving the lowest dose (250 < 0.01); this reflected the reduced weekly ppm) but there were significant increases food intake in these groups. in the relative liver weights of female rats Over the 13-week treatment period there receiving 500 ppm Cereclor S52 and above was a dose-related reduction in the body and in male rats receiving 2500 or 5000 weight gain of male rats receiving diets con- ppm. The relative kidney weights were sigtaining 500 ppm or more Cereclor S52, but nificantly increased in male and female rats the total food consumption of these groups receiving 5000 ppm of Cereclor S52 (Tawas also significantly less than control val- ble 4). ues. No effects attributable to the comElectron microscopy showed some evipound were seen on the food consumption dence of a dose-related proliferation of the or body weight gain of the female rats. In smooth endoplasmic reticulum in the hea subsidiary paired-feeding experiment patic cells of rats receiving 500 ppm and with groups of 12 male rats, control rats above of Cereclor S52 in their diet. No other were allowed the same weight of food as abnormalities were detected. Histochemical had been consumed by rats receiving un- examination revealed no abnormalities.
522
BIRTLEY
ET AL.
TABLE BODY
WEIGHT
GAIN
3
AND FOOD CONSUMPTION OF RATS TREATED SS2 CONTAINING STABILIZER IN DIET
Concentration of Cereclor S52 in diet (wm)
Mean total food consumed per cage of four rats 6s *SD)
WITH CERECLOR
Mean total body weight gain per cage of four rats 6s *SD)
Mean food conversion ratio (*SD)
Duration of treatment (weeks)
Number of rats per group
13 (subacute feeding toxicity study)
12
Male
0 500 2500 5000
8065 7530” 7286” 7407”
(2251) (2228) (2328) (?386)
1152 1056 961” 86y
(*98) (k68) (263) (k83)
7.0 7.1 7.6 8.5”
(20.7) (rtO.3) (kO.4) (20.8)
12
Female
0 500 2500 5000
5978 5766 5884 5949
(2178) (2302) (2145) (?248)
412 418 438 403
(-c25) (?37) (k60) (249)
14.5 13.8 13.4 14.8
(kO.9) (kl.1) (21.6) (k1.6)
12
Male
0 250
7092 (5253) 6893 ( t- 378)
1043 (k62) 1008 (a61)
6.8 (kO.3) 6.8 (kO.7)
12
Female
0 250
4976 (? 145) 5103 (2325)
367 (+22) 331 (246)
13.6 (kO.8) 15.4 (k2.2)
12
Male
0 500 w
2601 (294) 2435* (294) 2423* (289)
622 (?36) 597 (+42) 5546 (240)
4.2 (20.4) 4.1 (20.3) 4.4 (t-0.5)
4 (Paired-feeding study)
Sex
a Significance of difference from appropriate control mean value, p < 0.01. * Significance of difference from appropriate control mean value, p < 0.05. c Weight of diet offered daily restricted to that quantity eaten by 500 ppm group on previous day.
(b) Ninety-day feeding study in dogs with
Cereclor X52. The results of diet analyses showed adequate mixing of chlorinated paraffin with the food; achieved concentrations were similar to nominal values. All animals survived the treatment period and they remained clinically well throughout with no evidence of abnormal behavior. Treatment with Cereclor ,952 at any dose level had no effect on weight gain, hematological parameters, or urinalysis results. The only parameters which were affected by treatment were confined to the male dogs which received 100 mg/kg/day, namely statistically significant (p < 0.05) increases in serum alkaline phosphatase activity and liver weight-to-body weight ratio. There were no macroscopic or microscopic lesions which were attributable to
treatment, except the occurrence of some cloudy, pale, and enlarged hepatocytes in some dogs which received 30 and 100 mg/ kg/day. Electron microscopy revealed an increase in the smooth endoplasmic reticulum of hepatocytes in all dogs receiving 30 and 100 mglkg/day. Analysis of the number of hepatocyte nuclei per unit area suggested that the increase in liver weight was due to the increased number of cells rather than to cell enlargement, but this is not a precise means of distinguishing between these two possible responses. Histochemitally there were no abnormalities detected at any dose level. 5. TissueRetention and Elimination of Radioactive Cereclor S52 in Rats
Following dietary administration of [36C1]Cereclor S52 at either 0.4 or 40 ppm,
TOXICOLOGY
ORGAN-TO-BODY
Number of rats per group
WEIGHT
Sex
OF CHLORINATED
523
PARAFFINS
TABLE 4 RATS TREATED WITH CERECLOR S52 CONTAINING STABILIZER IN DIET FOR 13 WEEKS RATIO
IN
Mean organ-to-body weight ratio (g/100 g *SD)
Concentration of Cereclor S52 in diet (ppm)
Mean final body weight (g &SD)
Liver
Kidneys
12
Male
0 500 2500 5000
454 431 415” 409”
(?39) (+29) (238) (?39)
3.54 3.46 4.086 4.30*
(kO.36) (20.22) (?0.47) (kO.28)
0.58 0.59 0.64 0.67”
(20.09) (20.03) (%0.06) (kO.06)
12
Female
0 500 2500 5000
275 275 290 276
(217) (~21) (k26) (k23)
3.36 3.72* 4.07* 4.96*
(kO.26) (kO.31) (kO.44) (kO.42)
0.61 0.64 0.62 0.706
(eO.06) (kO.05) (kO.10) (20.07)
12
Male
0 250
448 (241) 442 (k30)
3.13 (?0.35) 3.37 (kO.42)
0.53 (kO.03) 0.53 (kO.07)
12
Female
0 250
250 (k26) 248 (k23)
3.31 (kO.40) 3.24 (kO.40)
0.59 (LO.04) 0.61 (kO.05)
u Significance of difference from corresponding * Significance of difference from corresponding
control, p < 0.05. control, p < 0.01.
equilibrium levels of radioactivity were established in liver and abdominal fat within 1 and 7 weeks, respectively. No radioactivity was detected in brain and adrenal glands. When the diet was changed from 0.4 ppm radioactive chlorinated paraffin to that containing none, it was estimated that the half-time for removal of radioactivity from abdominal fat was about 8 weeks (Table 5), and the level of radioactivity in the liver declined below the detection limit within 1 week. No attempt was made to characterize the radioactivity in any tissue. DISCUSSION Three grades of chlorinated paraffins, representative of both the range of structural types within this chemical family and of the grades produced in greatest tonnages, failed to show a positive response in either bacterial or mammalian cell test systems which, in combination, seem likely to detect substances which possess potential carcinogenic
or mutagenic
properties
(Fur-
chase et al., 1976). It is known that neither of these tests is capable of detecting certain substances, for example, chloroform, which may produce their carcinogenic effects by epigenetic mechanisms. It is not considered likely that chlorinated paraffins could exert such an effect, because it is improbable that any low molecular weight organochlorine moieties will be formed in viva as intermediates and which could conceivably cause a mutagenic or carcinogenic effect. Chlorinated paraffins are of very low acute oral toxicity and possess only slight skin and eye irritation properties. By extrapolation, they should be of low risk to. man in the event of ingestion or skin and eye contact and during nearly 40 years of chlorinated paraffin manufacture there is no record of any adverse clinical reactions among production staff who have been potentially exposed to these chemicals. From the various studies in which more than one type of chlorinated paraffin was tested, indications of differential toxicity
524
BIRTLEY
ET AL.
were sought. In terms of acute oral toxicity, skin irritancy, and eye irritancy there was evidence that the effects, although only slight, were more pronounced with the short carbon chain types. This observation may be explained in terms of a greater inherent toxicity in these types of chlorinated paraffin or that, since they are of lower molecular weight and viscosity than longer carbon chain varieties, they are simply more readily absorbed in order to exert the observed effect. Within the short carbon chain types, there was no real evidence that the extent of chlorination had any relationship to the nature or intensity of the toxic effects. The relative in vitro cytotoxic effect on baby hamster kidney cells of the four substances evaluated in the cell transformation test cannot be explained in terms of degree of chlorination or carbon chain length. Two features are clear, namely that there is at least almost an order of magnitude of difference between the L&, values of each chlorinated paraffin tested (suggesting that the differences are real) and that addition of stabilizer to Cereclor S52 enhances its toxicity to BHK cells by a factor of about 5. The difference in relative toxicity of the
various grade of chlorinated paraffin in in viva systems compared with that in an in vitro system serves to illustrate the difficulties in making comparisons between results obtained in the two types of system. The nature of the hepatic changes caused by Cereclor S52 following continuous administration in the diet to rats and dogs for 3 months is similar to that caused by certain other lipid-soluble compounds. Absence of any treatment-related enzyme histochemical or histopathological abnormalities in the liver suggests that the observed liver weight increases and the proliferation of smooth endoplasmic reticulum are adaptive responses due to stimulation of the synthesis of foreign compound metabolizing enzymes in this organ. In this respect, the rat seems to be as sensitive to Cereclor S52 as the dog, but the effects occurred in both species only at high dietary concentrations; no effects were detected by electron microscopy at dietary concentrations of 250 ppm and 10 mg/kg/day (equivalent to about 300 ppm) in rats and dogs, respectively. In order to assess the risk to man in terms of exposure levels, this information, coupled with the finding that Cereclor S52 was
TABLE THE
CONCENTRATION OF RADIOACTIVITY, PER GRAM TISSUE, IN THE LIVER AND
Dietary CO”Ceiltration of [“Cl] CWXlOI s52 (wm) 0.4
40
CALCULATED ABDOMINAL
Tissue
5 AS MICROGRAM FAT OF RATS FED
concentration
of Cereclor
EQUIVALENTS [36C1]C~~~~~~~
Tissue sample Liver Map” Abdominal fat MealP Range Liver Mean” Abdominal fat MCUI” Range
n Mean of three values. b Mean of three values,
feeding
S52
return
diet
S52 (Irglg) After
After
OF CERECLOR S52 IN DIET
test diet for (Weeks)
to untreated (Weeks)
1
2
3
4
5
6
7
8
9
10
1
0.08
0.07
0.10
0.07
0.07
0.06
0.06
0.12
0.09
0.09
0.01
0.0
0.14 O.ll0. I8
0.22 O.lS0.24
0.28 0.25% 0.33
0.30 0.280.33
0.30 0.280.31
0.38 0.320.43
0.38 0.3% 0.40
0.36 0.340.39
0.35 0.330.38
0.37 0.330.40
0.29 0.260.34
0.3 I 0.300.32
7
7
6
5
6
23 2223
30 2832
33 3134
39 3542
33 3234
7
13 1017
21 2023
but results
at 6 and Cl weeks after return
32 3034
to untreated
diet are the mean of two values.
3
6
8
0.22 -
0.19 -
TOXICOLOGY
OF CHLORINATED
PARAFFINS
525
found in rat abdominal fat (where the toxicity for the chlorinated paraffins and greatest tissue concentrations occurred) at that. they are unlikely to be mutagenic or concentrations which were not greater than carcinogenic. those in the diet, is of value. Campbell and McConnell (1980) found comparable conACKNOWLEDGMENTS centrations of chlorinated paraffins in human adipose tissue and certain foodstuffs, The authors thank Dr. T. F. McElligott, Mrs. S. E. providing direct evidence that at least in Moses, Dr. J. Styles, Mr. H. Bratt, Mr. G. H. Walker, Mr. M. J. L. Clapp, Dr. R. Gamer, Dr. D. A. Kinch, this respect, rats and man are comparable. Mr. M. Watson, and Mr. R. B. Wright for their With the limited data presented in Campbell’s paper, it is difficult to estimate the contributions to the work described in this paper. chlorinated partin concentration in total diet; a realistic upper estimate is 0.1 ppm REFERENCES which provides a several thousandfold AMES, B. N., MCCANN, J., AND YAMASAKI, E. (1975). safety factor between rats and man. AsMethods for detecting carcinogens and mutagens suming that man will respond in a similar with the Salmonella/mammalian-microsome mutaway to that seen in rats and dogs to the genicity test. Mural. Res. 31, 347-364. effects of chlorinated paraffins, and that CAMPBELL, I., AND MCCONNELL, G. (1980). Chloriman is more sensitive than these species nated paraffins in the environment. Environ. Sci. Technol., in press. to such effects, the safety margin is still CURLEY, A., BURSE, V. W., GRIM, M. E., JENNINGS, sufficiently large to enable one to conclude R. W., AND LINDER, R. E. (1971). Polychlorinated that the use of these chemicals presents little biphenyls: Distribution and storage in body fluids or no risk to man. and tissues of Sherman rats. Environ. Res. 4, Two important findings in the tissue reten481-495. tion studies in rats were that equilibrium DANIEL, J. W., AND GAGE, J. C. (1965). The absorption and excretion of butylated hydroxytoluene concentrations of radioactivity in abdominal (BHT) in the rat. Food Cosmet. Toxicol. 3,405-415. fat were similar to but never exceeded DOBBS, H. E. (1963). Oxygen flask method for the the dietary concentration of chlorinated assay of tritium, carbon-14 and sulfur-35 labelled paraffin, and that the levels of radioactivity compounds. Anal. Chem. 35, 783-786. in this tissue declined fairly rapidly after DRAIZE, J. H. (1959). Appraisal of the Safety of Chemicals in Food, Drugs and Cosmetics, pp. treatment stopped. These observations 46-59. Association of Food and Drug Officials of were made after feeding a diet containing a the United States, Washington, D.C. similar concentration of chlorinated paraf- KAY, J. H., AND CALANDRA, J. C. (1%2). Interpretafin to that estimated for man. Other organotion of eye irritation tests. J. Sot. Cosmet. Chem. chlorine compounds such as DDT (Laug 13, 281-289. LAUG, E. P., NELSON, A. A., FITZBURGH, 0. G., AND et al., 1950), Aldrin and Dieldrin (Quaife KUNZE, F. M. (1950). Liver cell alteration and DDT et al., 1967), and Aroclor 1254, a polystorage in the fat of the rat induced by dietary levels chlorinated biphenyl (Curley et al., 1971), of 1 to 50ppm DDT. J. Pharmacol. Exp. Ther. 98, have been shown to accumulate to con268-273. centrations in adipose tissue which are PURCHASE, I. F. H., LONGSTAFF, E., ASHBY, J., STYLES, J. A., ANDERSON, D., LEFEVRE, P. A., several times greater than the dietary conAND WESTWOOD, F. R. (1976). Evaluation of six centration fed to animals. This is an imshort term tests for detecting organic chemical portant difference between chlorinated carcinogens and recommendations for their use. paraffins and the other substances, and is an Nature (London) 264, 624-627. influential factor in the consideration that QUAIFE, M. L., WINBUSH, J. S., AND FITZHUGH, 0. G. (1967). Survey of quantitative relationships between chlorinated paraffins present a low potential ingestion and storage of aldrin and dieldrin in risk to man. The other factors supporting animals and man. Food Cosmet. Toxicol. 5, 39-50. this conclusion, which have been estab- STYLES, J. A. (1977). A method for detecting carlished from the investigations reported in cinogenic organic chemicals using mammalian cells in culture. Brit. J. Cancer 36, 558-563. this paper, are the low acute and subacute
AND
APPLIED
PHARMACOLOGY
The Toxicological ROBIN DUNCAN
Imperial
Effects
54,
514-525 (1980)
of Chlorinated
Paraffins
in Mammals
NICHOLAS BIRTLEY, DAVID MICHAEL CONNING,’ JOHN WYNFORD DAVID MACKAY FERGUSON,~ ERIC LONGSTAFF, AND ANDREW ALEXANDER BUCHANAN SWAN
Chemical
Industries
Limited, Central Toxicology Laboratory, Cheshire SK10 4TJ, England
Received
September
4, 1979; accepted
March
Alderley
Park,
DANIEL,~
Macclesjield,
28, 1980
The Toxicology of Chlorinated Paraffins in Mammals. BIRTLEY, R. D. N., CONNING, D. M., J. W., FERGUSON, D. M., LONGSTAFF, E., AND SWAN, A. A. B. (1980). Toxicol. Appl. Pharmacol. 54, 514-525. Chlorinated paraffins are a group of chemicals derived from n-paraffins containing between 10 and 30 carbon atoms which are chlorinated to between 40 and 70% (w/w). Representative grades were tested for acute oral toxicity and skin irritancy in rats, eye irritancy in rabbits, and potential mutagenic effects in two in vitro tests. The effects of one grade (14-17 carbon atoms, 52% chlorinated) which is manufactured in high-tonnage quantities, was evaluated in a 90-day feeding study in rats and dogs, and its tissue retention and elimination was determined in the rat. Chlorinated paraffins are of low acute oral toxicity. Slight, reversible skin and eye irritations were produced by shorter carbon chain derivatives only and no type showed any evidence of mutagenic potential. Subacute administration of 500, 2500, and 5000 ppm reduced diet palatability to rats. Increases in SGPT or SAP levels, liver weight, and smooth endoplasmic reticulum in hepatocytes were seen in rats and dogs: “no-effect” diet levels were 250 and 300 ppm, respectively. Chlorinated paraffin accumulates primarily in the abdominal fat of rats at concentrations which never exceed those in diet (unlike certain other chlorinated organic compounds); its half-life in abdominal fat is about 8 weeks, when untreated diet is offered. On the currently available evidence, in view of their slight toxicity and the low human dietary levels in comparison to the maximum “no-effect” levels in rats and dogs, it is concluded that chlorinated paraffins present no toxicological risk to man despite their continued manufacture and widespread low-level occurrence in the environment. DANIEL,
Chlorinated paraffins are a large group of industrially important chemical types, the extremes of which are n-paraffins of 10 and 30 carbon atoms chlorinated to 40 and 70% by weight. There are many grades of chlorinated paraffin, each of which is a complex mixture of compounds and iso’ Present address: British Industrial Biological Research Association, Woodmansteme Road, Carshalton, Surrey SM5 4DS, England. * Present address: Life Science Research, Stock, Essex CM4 9PE, England. 3 Present address: Imperial Chemical Industries Americas Inc., Wilmington, Del. 19897. 0041-008x/80/090514-12$02.00/0 Copyright All rights
D 1980 by Academic Press, Inc. of reproduction in any form reserved.
mers representing a small spectrum within the ranges of carbon chain length and extent of chlorination. Each grade has specific physical and chemical properties which render them useful in such widespread applications as plasticisers in plastics such as polyvinyl chloride, extreme pressure additives for gear and cutting oils, fire-retardant additives, paints, and electrical insulators. They have been in use for up to 40 years. There are no reports in the literature of the toxicological effects of chlorinated paraffins in mammals. The studies reported here describe the results obtained in two in 514
TOXICOLOGY
OF CHLORINATED
tests which predict mutagenic potential and in mammalian acute toxicity and irritancy tests with a variety of chlorinated paraffin grades which represent the spectrum of chemical types and which are manufactured in significant quantities. Also, results of subacute toxicity studies in rats and dogs together with tissue retention and elimination studies in rats are described for one grade of chlorinated paraffin which is manufactured in large quantities. These are the studies which have been completed to date in a major ongoing program designed to evaluate the toxicological potential of chlorinated paraffins.
vitro
METHODS 1. Salmonella typhimurium (Ames test)
Reverse Mutation
Assay
The following chlorinated paraffin grades were evaluated in this test: Cereclor’ S52 (14- to 17-carbon n-paraffins, chlorinated to 52% on average), this grade was evaluated without and with 0.2% (w/v) epoxidized vegetable oil added as stabilizer; Cereclor’ 42 (20- to 30-carbon n-paraffins, chlorinated to 42% on average); Cereclor’ 50LV (lO- to 13-carbon n-paratlins, chlorinated to 50% on average). The method used was essentially the same as that described by Ames et al. (1975). Each grade was tested at five dose levels (2500, 500, 100, 20, and 4 pg per plate) on each of 5 consecutive days, and each dose level was tested in duplicate. Four strains ofSalmonella typhimurium were used throughout, namely TA1535, TA1538, TAlOO, and TA98, both with and without Aroclor-induced rat liver postmitochondrial supernatant (S-9 mix). 1,3-Propanesultone was used as positive control substance for TA1535 and TAlOO strains while 2-acetylaminofluorene was used for the other two strains. Dimethyl sulfoxide was used as vehicle for all test substances and as the negative control substance.
’ The name “Cereclor” is a trademark, the property of Imperial Chemical Industries Limited, for the chlorinated paraffin product range. The letters and digits identify further the specific grades. All chlorinated paraffins were supplied by ICI Mond Division, PO Box 8, The Heath, Runcorn, Cheshire WA7 4QD, England.
PARAFFINS
2. Cell Transformation
515 Test
The same three grades of chlorinated paraffin which were evaluated in the S. typhimurium test were also tested here. The procedure was identical to that described by Styles (1977). Five concentrations of each test compound in the final mixture were used, namely 2500,250, 25,2.5, and 0.25 &ml. 2-Acetylaminofluotene (1250 &ml) was the positive control substance. 3. Acute Toxicity and Irritancy Studies (a) Acute oral toxicity to rats. A single oral dose of a chlorinated paraffin, either as the material as supplied or in a vehicle such as olive oil, was administered to groups of at least three female and/or male Alderley Park Wistar rats.* In any one experiment, the body weights of all animals were within 50 g, but the weights of the animals for all studies were in the range of 125-250 g. They were deprived of food for 16 hr prior to dosing, and then allowed food and water ad [ibitum during the remainder of the experiment. The dose levels used were between 0.5 and 10 g/kg body weight. The animals were regularly observed for abnormal clinical signs for up to 14 days after treatment and an autopsy was performed at the end of the observation period (or at death if this was sooner). Occasionally, certain organs or tissues which were considered to have been affected by treatment were processed by standard histological techniques and sections were examined microscopically. (b) Skin irritancy evaluation. A total of up to six applications of each grade tested was made to the shorn back of each of three female rats* (125-250 g body wt) on alternate 24-hr periods. The volume applied was generally 0.1 ml, either as the neat chlorinated pa&tin or as a concentrated solution in a nonirritant vehicle. The treated skin was kept covered with an occlusive dressing while in contact with the test agents, and at the end of each 24-hr contact period the skin was washed once with a weak detergent solution. The rats were housed singly during the experimental period and allowed free access to food and water. Skin reaction to treatment was subjectively assessed 24 hr after removing each application and on some occasions immediately after washing the skin. The severity of erythema, desquamation, peeling, necrosis, etc., were recorded for each rat on all occasions. If severe reactions occurred in any animal before the scheduled experimental period ended, the testing in that animal was terminated prematurely. In certain instances such animals and some which completed the * Supplied by the Animal Breeding Unit, Imperial Chemical Industries Ltd, Alderley Park, Macclesfield, Cheshire, England.
516
BIRTLEY
ET AL.
TABLE NUMBER
OF SAMPLES
1
OF CHLORINATED
THREE
TYPES
PARAFFINS
OF ACUTE
EVALUATED
TOXICITY
IN EACH
OF
TEST
Extent of chlorination
of n-paraffin (by weight) 51-60%
41-50%
61-70%
Number of carbon atoms in n-pa&in chain
AOb
SIC
EI”
A0
SI
EI
A0
SI
El
10-13
3
8
0
0
0
6 9
0 6
9 0
3
0
2 8
11
14-17
20-30
2
1
0
1
1
0
2
00 2
2
a AO, Acute oral toxicity. b SI, Skin irtitancy evaluation. c EI, Eye irritancy evaluation. d Each sample of chlorinated paraffin is derived from a mixture of n-paraffins containing the range of numbers of carbon atoms shown, but it is chlorinated to a specific percentage by weight. The table shows the number of samples evaluated in each type of test whose percentage chlorination falls within the arbitrary ranges shown. duration of the experiment were subjected to autopsy procedures and any abnormalities were noted. (c) Eye irritancy evaluarion. A single application (usually 0.1 ml) of undiluted chlorinated paraffin was instilled into the conjunctival sac of one eye of each of three New Zealand White rabbit? (2.5-3.5 kg body wt) of either sex. The other eye served as a contralateral control. The rabbits were housed singly and allowed food and water ad l&turn during the experimental period. The irritancy induced was scored 1 hr after instillation then at daily intervals up to 7 days afterward, by the method of Draize (1959) as modified by Kay and CaIlandra (1962). A subjective assessment of initial pain reaction was made in some experiments. Any animal seen to be unduly stressed as a result of the treatment was killed. The number of grades of chlorinated paraffin, classified broadly according to the number of carbon atoms in the n-paraffin chain and the extent of chlorination by weight, which were evaluated in acute toxicity and irritancy tests are summarized in Table 1. Identical grades, which contain small amounts of different types of stabilizer, have been counted as separate test compounds.
4. Subacute
Toxicity
(a) Ninety-day feeding containing stabilizer.
Studies study
in rats with Cereclor
S52-
Specific pathogen-free, Wistarderived rats (body weight, 160- 180 g) were randomly selected from a larger population* and were housed in wire mesh cages suspended on racks. Groups of
48 rats (24 males, 24 females) were housed four per cage and maintained for 90 days on diets containing 500, 2500, or 5000 ppm Cereclor ,852~containing stabilizer; the diet of a similar control group of 48 rats contained the vehicle, corn oil only. Food3 and water were available ad libitum. At 6 weeks, a further group of 48 rats whose diet contained 250 ppm Cereclor S52 with stabilizer was introduced into the experiment together with an additional control group of 12 male and 12 female rats; these rats were treated in an identical manner to those on the main study. The concentration and distribution of the chlorinated paraffin in each batch of diet were checked by chemical analysis. Each rat was observed daily throughout the treatment period for clinical abnormalities. The body weight of each rat was recorded initially and at weekly intervals together with the food consumption per cage. Samples of blood were taken from 10 males and 10 females from each group preexperimentally, at 6 weeks and terminally for individual determination of haemoglobin concentration, packed cell volume, mean corpuscular diameter. reticulocyte count, total and differential white cell counts and platelet counts. Clotting function tests (prothrombin index and kaolincephalin time) were performed terminally. Plasma alkaline phosphatase and glutamic pyruvate transaminase activities were assayed in samples from four males and four females from each treatment group preexperimentally, after 6 weeks and terminally. At the end of the 90-day period all animals were killed with a dose of volatile anaesthetic and an im3 Oakes Ltd., Congleton, Cheshire,
England.
TOXICOLOGY
OF CHLORINATED
mediate full postmortem examination made. The liver, kidneys, adrenals, lung, heart, and spleen from half of the animals of each sex in each group were weighed and organ-to-body weight ratios calculated. The following organs were examined microscopically after preservation in form01 corrosive, processing to paraffin wax blocks and hematoxylin and eosinstained sections (5 pm) mounted on slides: liver, kidney, spleen, heart, lung, adrenal, gonads, thymus, thyroid, pancreas, stomach, duodenum, jejunum, cecum, ileum, colon, salivary gland, and mesenteric lymph nodes. The brain (cerebrum, cerebellum, and pans) and spinal cord were fixed in 10% form01 saline prior to processing and sectioning. Sections of liver from three male and three female animals from each group, which had been deprived of food overnight, were preserved for electron microscopic examination. Sections of liver, kidney, spleen, adrenals, pancreas, lung, gonad, stomach, duodenum, ileum, and colon from similar groups of animals were preserved in liquid nitrogen for histochemical analysis of certain enzymes. fh) Ninety-day feeding study containing stabilizer. Beagle
in dogs with Cereclor
S52
dogs from an inbred strain’ (body weight, lo-14 kg), which had been immunized against distemper and hardpad were housed in individual pens and, prior to experimental assignment, received two doses of piperazine adipate5 to exclude nematode infection. Their main meal, offered at noon each day, comprised 45 g of a meat preparation” and 226 g of a dried pelleted diet:7 a dog biscuits weighing 56 g was offered each afternoon. Each animal had its own automatic drinking fountain. Cereclor SS2-containing stabilizer was diluted with corn oil and mixed in a ball mill for 24 hr. The concentration was adjusted to allow dosage at the rate of 1 ml/kg body wt per day, and the resultant solution was syringed into and thoroughly mixed with the main meal. Four male and four female beagles at each dose level were dosed daily for 90 days with 0, 10, 30. or 100 mgikg body wt of Cereclor S52. The distribution and final concentration of chlorinated paraffin in diets were checked by chemical analysis. Each animal was observed daily for gross clinical effects; detailed examinations were made preexperimentally and terminally. The body weight of each dog was determined preexperimentally and then weekly throughout the experiment. Samples of blood were taken from each dog pre’ Epivax, Burroughs Wellcome and Co., London. j Coopane, Cooper, McDougall and Robertson Ltd., Berkhamsted, Hertfordshire. England. ’ Kennomeat, Scottish Animal Products, Barrhead, Scotland. ’ Kennel Kernals. James and Co.. Hungerford, Berkshire, England. ’ Winalot, Spillers Ltd., London, England.
517
PARAFFINS
experimentally and after 42 and 90 days of treatment for determination of hemoglobin, packed cell volume, mean cell hemoglobin concentration, mean cell diameter, reticulocyte count, leukocyte count (total and differential), platelet count and clotting function (kaolin-cephalin time and prothrombin index). Bone marrow cytology was investigated by needle biopsy from the iliac crest between Days 40 and 50 and terminally. Also, for all dogs in each group, blood glucose, blood urea, plasma alkaline phosphatase, glutamic pyruvic transaminase, sodium, and potassium were determined at the same times as the hematology parameters were measured. Urinalysis (pH, specific gravity, protein, glucose, and bilirubin) was undertaken in samples of urine obtained overnight from each dog preexperimentally and terminally. Electrocardiography was also performed preexperimentally and terminally. At the end of the 90-day feeding period all dogs were killed by intravenous administration of a lethal dose of pentabarbital sodium and a full postmortem examination was made. Heart, liver, kidneys, adrenals. spleen, thyroids, testes, lungs, and brain were weighed and organ-to-body weight ratios calculated. Representative pieces of tissue were taken from the pituitary, salivary gland, thyroid, thymus, heart, lung, aorta, stomach, duodenum, jejunum, ileum, colon, liver, spleen, kidney, bladder, adrenal, lymph nodes, ovary and uterus, testis, and epididymis; fixed, processed into paraffin wax, 5-pm sections stained with hematoxylin and eosin and examined microscopically. Samples of brain, spinal cord and sciatic nerve were taken from all groups. although only those from the controls and highest dose group were processed and examined. Samples were also taken from the major lobes of the livers of all animals and processed for examination in the electron microscope. Similar samples were frozen in liquid nitrogen and processed for a range of enzyme histochemical reactions: in addition the sections were analyzed for the numbers of nuclei per unit area as a measure of cell size. 5. Tissue Cereclor
Retention and S52 in Rats
Elimination
of Radioactive
lS”Cl]Cereclor S52 was synthesized by chlorination to 52% (w/w) of commercial C,,-C,, n-paraffin feedstock with [3”Cl]chlorine which had been generated from a solution of sodium [36Cl]chloride (41.7 &i/ml)Y by reaction with hydrogen peroxide and oleum; the specific activity of the purified product was 0.36 mCi/g. Male Wistar rats (180-200 g body wt) of the Alderley Park Strain2 were used throughout. Food3 and y Radiochemical shire, England.
Centre, Amersham, Buckingham-
518
BIRTLEY
ET AL.
TABLE THE MEAN DAILY NUMBER OF CHLORINATED PARAFFIN, FOUR STRAINS OF Salmonella
2
OF REVERTANT COLONIES PER PLATE FOR EACH DOSE TESTED OF THREE GRADES Two POSITIVE CONTROL SUBSTANCES, AND A VEHICLE CONTROL WITH EACH OF typhimurium IN THE PRESENCE AND ABSENCE OF RAT LIVER S9-MIX
Strain of S. typhimurium TA1535 Test substance
Dose ( wdplate)
+
without (-) or with (+) SPmix
TA1538 -
TAlOO
TA98
+
-
+
-
+
-
Cereclor S52 without added stabilizer
2500 500 100 20 4
11 18 29 16 25
9 9 8 8 8
22 32 12 58 63
6 7 4 6 7
53 46 43 42 34
48 42 45 46 39
12 15 15 18 14
14 9 9 11 10
Cereclor S52 with added stabilizer
2500 500 100 20 4
16 24 18 15 21
13 9 8 9 10
24 10 11 14 13
8 9 8 10 10
50 37 42 41 42
31 30 25 35 33
20 13 11 13 8
10 10 12 9 11
Cereclor 42
2500 500 100 20 4
12 14 14 12 13
8 7 8 9 7
14 12 15 14 15
8 6 8 9 6
36 35 30 39 33
35 26 32 33 30
19 14 12 15 11
7 10 9 6 8
Cereclor 50LV
2500 500 100 20 4
10 6 11 8 6
7 7 11 8 8
8 11 14 15 8
10 7 12 12 7
37 38 33 26 37
34 29 26 24 28
11 11 9 14 10
7 7 7 6 7
1,3-Propane sultone
500 100 20
489 287 148
340 219 161
2-Acetylamino fluorene
500 100 20
213 251 208
9 10 13
Dimethylsulfoxide (vehicle control)
-
30
10
water were available ad libirum mental periods.
38
during the experi-
(a) Tissue retention and elimination studies. The retention of radioactivity was investigated in two groups of rats which were maintained on diets containing either 0.4 or 40 ppm [30Cl]Cereclor S52. The required amount of Cereclor S52 was dissolved in corn oil which was then added to malt extract (18 parts) and water (5 parts) and the whole mixed mechanically with stock diet3 (77 parts). Animals were fed the experimental diet ad libitum for either 8 (40 ppm) or 10 weeks (0.4 ppm). Groups of three animals were killed
11
546 346 277 280 188 198 165 148 118
8 9 8
20
10
74
60
at weekly intervals. After 10 weeks, the surviving animals which had been maintained on the 0.4-ppm diet were returned to a control diet and three rats were killed at varying times during the subsequent 8 weeks. Abdominal fat, liver, brain, and adrenal glands were removed from all rats immediately after death and the quantity of radioactivity in them was measured. (b) Analytical
methods for measurement
of radioactivity
Abdominal fat and brain were macerated in p-dioxane (20 ml) and liver was homogenized in acetone (20 ml). The suspensions were centrifuged and the supernatant solutions retained. The radioactivity in these
TOXICOLOGY
OF CHLORINATED
PARAFFINS
519
fluorene caused greater than five times the background transformation frequency of approximately 50 per lo6 surviving cells, showing that the test was working satisfactorily. None of the concentrations of the chlorinated paraffins tested caused the cell transformation frequency to exceed background values, even at the LCsO value. RESULTS The relative lethality of the four test subI. Salmonella typhimurium Reverse Mutation stances to baby hamster kidney cells is of Assay (Ames Test) interest. The LC,,, values for Cereclor SOLV, 42, S52 with stabilizer, and S52 without stabiThe results are summarized in Table 2. lizer were estimated from dose-lethality There was no indication that chlorinated curves to be approximately 1.3,36,228, and paraffins were toxic to any of the four tester 1250 pg/ml, respectively. strains of S. typhimurium at doses up to 2500 pg per plate. The test system was shown to 3. Acute Toxicity and Irritancy Tests be working satisfactorily as dose-response relationships were established for 1,3-pro(a) Acute oral toxicity. Out of all the difpanesultone, and activation of 2-acetylferent grades of chlorinated paraffins tested aminofluorene in the presence of S9-mix (Table l), clinical signs of toxicity were conwas evident; both positive control sub- fined mainly to rats which received a high stances displayed unequivocal mutagenic dose (4 g/kg body wt or above) of chloriproperties in this test. Using the criterion nated paraffins with lo-13 carbon atoms that the ratio of the test to negative control in the n-paraffin chain. Observations of response should be at least 2.0 to provide toxicity which were commonly made were evidence of a mutation, it can be seen from nonspecific and included piloerection, Table 2 that no such evidence exists for muscular incoordination and urinary and any of the chlorinated paraffin grades fecal incontinence. These animals usually tested. The test response was greater than recovered completely within 7 days of treatdouble the negative control value when the ment. The intensity and nature of these two lowest doses of Cereclor S52 without responses was generally independent of the added stabilizer were evaluated with the chlorine content of the substances tested. TA1538 strain in the presence of S9-mix. Thus the acute oral LD,, of all grades This is considered to be an anomalous find- tested exceeded 4 g/kg and in some cases ing since, in the absence of evidence for a 10 g/kg. direct toxic action of the chlorinated parThe few abnormalities observed at necaffin on the cells, a dose-response rela- ropsy and on microscopic examination of tionship would be expected if a real mutatissue sections were generally confined to genic effect was being exerted. Furtherthose rats which showed signs of toxicity. more, no indication of a mutagenic effect Occasional inflammation of the gastric muwas seen in any strain treated with Cereclor cosa was observed, indicating a localized S52 which contained stabilizer. irritant action following administration of some chlorinated paraffins. Histopathological abnormalities were confined to the 2. Cell Transformation Test liver (hepatocellular vacuolation and ocThe results are presented graphically in casional necrotic foci) and the kidney (cloudy swelling of some inner cortical cells). Fig. 1. In each experiment, 2-acetylaminoextracts was measured using an Intertechnique SL 30 liquid scintillation spectrometer as described by Daniel and Gage (1965). The adrenals and samples of airdried solvent-insoluble material from other tissues were combusted in an atmosphere of oxygen, and the [36Cl]chlorine absorbed and counted using a modification of the method of Dobbs (1963).
520
BIRTLEY
ET AL.
Ifqy %surYl”ors I
1.100
iI
1.100
: :
900
:
I : :
900.
: I
: 700
I
700
I
500.
i :
300.
: I I
:
Wansformants per 10’ survivors
:
500
tronsformonts per lo* survivors
:
I 100 ! .----------__--__________ .----------____________,
looI 0
0.25
-------------,--‘,,,,,,,------
100 ’ 2.5
25
Concentmtion
250
2% lo
0
“g/ml
0.25
2.5
25
Conwntmtion
4 2500
250
us/ml
I. 1.100 I
i I
I transformants per 106 survivors
500
i tranrformants par lo’ survivors
:
I
0
i
0.25
1.5
Concsntmtion
25
250
lo
ug/ml
500
0
0.25
2.5
Concontrotion
25
250
2!
0
ug/ml
FIG. 1. Cell transformation test. Survival and transformation frequency of baby hamster kidney cells exposed to various concentrations of Cereclor 50LV (top left), Cereclor 42 (top right) Cereclor S52 with added stabilizer (bottom left), and Cereclor S52 without added stabilizer (bottom right). The transformation frequency is measured at the LC,,; it should exceed 2.50 per 106 survivors (five times the background frequency) for a potential carcinogenic effect to be established.
(b) Skin irritancy evaluation. Under the stringent conditions of this test, most chlorinated paraffins showed some degree of skin irritation. A mild skin irritancy response was confined to some of the grades derived from n-paraffins containing lo- 13 and 14- 17 carbon atoms; the response was generally independent of the degree of chlori-
nation. The occasional responses which were of moderate intensity were produced by the C,,-,, n-paraffins chlorinated to 70%. The mild. erythema and desquamation responses were usually established by the third application, but subsequently the condition did not worsen and often improved. It is possible that the irritancy response
TOXICOLOGY
OF CHLORINATED
was partly due in some cases to the presence of stabilizer in the test substance. All the longer carbon chain chlorinated paraffins were nonirritant. In experiments where autopsy was performed at the end of the treatment period, no evidence of systemic toxicity could be found. (c) Eye irritancy evaluation. Of the few grades evaluated in this test only mild, transient eye irritation was seen, and this was confined to the short carbon chain n-paraffins. Specifically, slight redness of the conjunctiva lasted no more than l-2 days; no abnormalities were noted in any other ocular structures and no pain response was observed in any animal.
PARAFFINS
521
limited quantities of the diet containing 500 ppm Cereclor S52. Over a period of 28 days the food conversion ratios of these two groups were comparable to that of a group fed control diet ad libitum indicating that the reduction in body weight gain in the main experiment was related to reduced palatability of the diets containing up to 2500 ppm Cereclor S52. From the increased food conversion ratio seen in male rats receiving 5000 ppm in diet, an adverse effect on weight gain is also suspected (Table 3). The results of the haematological investigation showed no abnormality attributable to the test compound, although male rats which received 5000 ppm Cereclor S52 showed a reduction in kaolin-cephalin index which was not statistically significant. A marked decline in alkaline phospha4. Subacute Toxicity Studies tase activity over the test period was seen in all groups, including controls. The se(a) Ninety-day feeding study in rats with rum glutamic pyruvate transaminase acCereclor S52. Diet analysis showed adequate tivity also showed a tendency to decline mixing of chlorinated paraffin with the except in male rats receiving the highest food and the achieved concentrations were dose where it increased somewhat. similar to theoretical values. There were At gross postmortem examination the no deaths during the course of the experionly specific abnormality revealed was a ment and no clinical abnormalities; the be- tendency towards congestion of the kidney haviour of all groups appeared normal. with increasing dietary concentration of The group mean body weight of treated Cereclor S52. Microscopically there were male rats was significantly lower than no abnormalities that could be attributed that of the control rats from Week 4 to the test compound. No abnormalities in onward at 500 ppm (p < 0.05) and from organ-to-body weight ratio were observed Week 1 onward at 2500 and 5000 ppm (p in animals receiving the lowest dose (250 < 0.01); this reflected the reduced weekly ppm) but there were significant increases food intake in these groups. in the relative liver weights of female rats Over the 13-week treatment period there receiving 500 ppm Cereclor S52 and above was a dose-related reduction in the body and in male rats receiving 2500 or 5000 weight gain of male rats receiving diets con- ppm. The relative kidney weights were sigtaining 500 ppm or more Cereclor S52, but nificantly increased in male and female rats the total food consumption of these groups receiving 5000 ppm of Cereclor S52 (Tawas also significantly less than control val- ble 4). ues. No effects attributable to the comElectron microscopy showed some evipound were seen on the food consumption dence of a dose-related proliferation of the or body weight gain of the female rats. In smooth endoplasmic reticulum in the hea subsidiary paired-feeding experiment patic cells of rats receiving 500 ppm and with groups of 12 male rats, control rats above of Cereclor S52 in their diet. No other were allowed the same weight of food as abnormalities were detected. Histochemical had been consumed by rats receiving un- examination revealed no abnormalities.
522
BIRTLEY
ET AL.
TABLE BODY
WEIGHT
GAIN
3
AND FOOD CONSUMPTION OF RATS TREATED SS2 CONTAINING STABILIZER IN DIET
Concentration of Cereclor S52 in diet (wm)
Mean total food consumed per cage of four rats 6s *SD)
WITH CERECLOR
Mean total body weight gain per cage of four rats 6s *SD)
Mean food conversion ratio (*SD)
Duration of treatment (weeks)
Number of rats per group
13 (subacute feeding toxicity study)
12
Male
0 500 2500 5000
8065 7530” 7286” 7407”
(2251) (2228) (2328) (?386)
1152 1056 961” 86y
(*98) (k68) (263) (k83)
7.0 7.1 7.6 8.5”
(20.7) (rtO.3) (kO.4) (20.8)
12
Female
0 500 2500 5000
5978 5766 5884 5949
(2178) (2302) (2145) (?248)
412 418 438 403
(-c25) (?37) (k60) (249)
14.5 13.8 13.4 14.8
(kO.9) (kl.1) (21.6) (k1.6)
12
Male
0 250
7092 (5253) 6893 ( t- 378)
1043 (k62) 1008 (a61)
6.8 (kO.3) 6.8 (kO.7)
12
Female
0 250
4976 (? 145) 5103 (2325)
367 (+22) 331 (246)
13.6 (kO.8) 15.4 (k2.2)
12
Male
0 500 w
2601 (294) 2435* (294) 2423* (289)
622 (?36) 597 (+42) 5546 (240)
4.2 (20.4) 4.1 (20.3) 4.4 (t-0.5)
4 (Paired-feeding study)
Sex
a Significance of difference from appropriate control mean value, p < 0.01. * Significance of difference from appropriate control mean value, p < 0.05. c Weight of diet offered daily restricted to that quantity eaten by 500 ppm group on previous day.
(b) Ninety-day feeding study in dogs with
Cereclor X52. The results of diet analyses showed adequate mixing of chlorinated paraffin with the food; achieved concentrations were similar to nominal values. All animals survived the treatment period and they remained clinically well throughout with no evidence of abnormal behavior. Treatment with Cereclor ,952 at any dose level had no effect on weight gain, hematological parameters, or urinalysis results. The only parameters which were affected by treatment were confined to the male dogs which received 100 mg/kg/day, namely statistically significant (p < 0.05) increases in serum alkaline phosphatase activity and liver weight-to-body weight ratio. There were no macroscopic or microscopic lesions which were attributable to
treatment, except the occurrence of some cloudy, pale, and enlarged hepatocytes in some dogs which received 30 and 100 mg/ kg/day. Electron microscopy revealed an increase in the smooth endoplasmic reticulum of hepatocytes in all dogs receiving 30 and 100 mglkg/day. Analysis of the number of hepatocyte nuclei per unit area suggested that the increase in liver weight was due to the increased number of cells rather than to cell enlargement, but this is not a precise means of distinguishing between these two possible responses. Histochemitally there were no abnormalities detected at any dose level. 5. TissueRetention and Elimination of Radioactive Cereclor S52 in Rats
Following dietary administration of [36C1]Cereclor S52 at either 0.4 or 40 ppm,
TOXICOLOGY
ORGAN-TO-BODY
Number of rats per group
WEIGHT
Sex
OF CHLORINATED
523
PARAFFINS
TABLE 4 RATS TREATED WITH CERECLOR S52 CONTAINING STABILIZER IN DIET FOR 13 WEEKS RATIO
IN
Mean organ-to-body weight ratio (g/100 g *SD)
Concentration of Cereclor S52 in diet (ppm)
Mean final body weight (g &SD)
Liver
Kidneys
12
Male
0 500 2500 5000
454 431 415” 409”
(?39) (+29) (238) (?39)
3.54 3.46 4.086 4.30*
(kO.36) (20.22) (?0.47) (kO.28)
0.58 0.59 0.64 0.67”
(20.09) (20.03) (%0.06) (kO.06)
12
Female
0 500 2500 5000
275 275 290 276
(217) (~21) (k26) (k23)
3.36 3.72* 4.07* 4.96*
(kO.26) (kO.31) (kO.44) (kO.42)
0.61 0.64 0.62 0.706
(eO.06) (kO.05) (kO.10) (20.07)
12
Male
0 250
448 (241) 442 (k30)
3.13 (?0.35) 3.37 (kO.42)
0.53 (kO.03) 0.53 (kO.07)
12
Female
0 250
250 (k26) 248 (k23)
3.31 (kO.40) 3.24 (kO.40)
0.59 (LO.04) 0.61 (kO.05)
u Significance of difference from corresponding * Significance of difference from corresponding
control, p < 0.05. control, p < 0.01.
equilibrium levels of radioactivity were established in liver and abdominal fat within 1 and 7 weeks, respectively. No radioactivity was detected in brain and adrenal glands. When the diet was changed from 0.4 ppm radioactive chlorinated paraffin to that containing none, it was estimated that the half-time for removal of radioactivity from abdominal fat was about 8 weeks (Table 5), and the level of radioactivity in the liver declined below the detection limit within 1 week. No attempt was made to characterize the radioactivity in any tissue. DISCUSSION Three grades of chlorinated paraffins, representative of both the range of structural types within this chemical family and of the grades produced in greatest tonnages, failed to show a positive response in either bacterial or mammalian cell test systems which, in combination, seem likely to detect substances which possess potential carcinogenic
or mutagenic
properties
(Fur-
chase et al., 1976). It is known that neither of these tests is capable of detecting certain substances, for example, chloroform, which may produce their carcinogenic effects by epigenetic mechanisms. It is not considered likely that chlorinated paraffins could exert such an effect, because it is improbable that any low molecular weight organochlorine moieties will be formed in viva as intermediates and which could conceivably cause a mutagenic or carcinogenic effect. Chlorinated paraffins are of very low acute oral toxicity and possess only slight skin and eye irritation properties. By extrapolation, they should be of low risk to. man in the event of ingestion or skin and eye contact and during nearly 40 years of chlorinated paraffin manufacture there is no record of any adverse clinical reactions among production staff who have been potentially exposed to these chemicals. From the various studies in which more than one type of chlorinated paraffin was tested, indications of differential toxicity
524
BIRTLEY
ET AL.
were sought. In terms of acute oral toxicity, skin irritancy, and eye irritancy there was evidence that the effects, although only slight, were more pronounced with the short carbon chain types. This observation may be explained in terms of a greater inherent toxicity in these types of chlorinated paraffin or that, since they are of lower molecular weight and viscosity than longer carbon chain varieties, they are simply more readily absorbed in order to exert the observed effect. Within the short carbon chain types, there was no real evidence that the extent of chlorination had any relationship to the nature or intensity of the toxic effects. The relative in vitro cytotoxic effect on baby hamster kidney cells of the four substances evaluated in the cell transformation test cannot be explained in terms of degree of chlorination or carbon chain length. Two features are clear, namely that there is at least almost an order of magnitude of difference between the L&, values of each chlorinated paraffin tested (suggesting that the differences are real) and that addition of stabilizer to Cereclor S52 enhances its toxicity to BHK cells by a factor of about 5. The difference in relative toxicity of the
various grade of chlorinated paraffin in in viva systems compared with that in an in vitro system serves to illustrate the difficulties in making comparisons between results obtained in the two types of system. The nature of the hepatic changes caused by Cereclor S52 following continuous administration in the diet to rats and dogs for 3 months is similar to that caused by certain other lipid-soluble compounds. Absence of any treatment-related enzyme histochemical or histopathological abnormalities in the liver suggests that the observed liver weight increases and the proliferation of smooth endoplasmic reticulum are adaptive responses due to stimulation of the synthesis of foreign compound metabolizing enzymes in this organ. In this respect, the rat seems to be as sensitive to Cereclor S52 as the dog, but the effects occurred in both species only at high dietary concentrations; no effects were detected by electron microscopy at dietary concentrations of 250 ppm and 10 mg/kg/day (equivalent to about 300 ppm) in rats and dogs, respectively. In order to assess the risk to man in terms of exposure levels, this information, coupled with the finding that Cereclor S52 was
TABLE THE
CONCENTRATION OF RADIOACTIVITY, PER GRAM TISSUE, IN THE LIVER AND
Dietary CO”Ceiltration of [“Cl] CWXlOI s52 (wm) 0.4
40
CALCULATED ABDOMINAL
Tissue
5 AS MICROGRAM FAT OF RATS FED
concentration
of Cereclor
EQUIVALENTS [36C1]C~~~~~~~
Tissue sample Liver Map” Abdominal fat MealP Range Liver Mean” Abdominal fat MCUI” Range
n Mean of three values. b Mean of three values,
feeding
S52
return
diet
S52 (Irglg) After
After
OF CERECLOR S52 IN DIET
test diet for (Weeks)
to untreated (Weeks)
1
2
3
4
5
6
7
8
9
10
1
0.08
0.07
0.10
0.07
0.07
0.06
0.06
0.12
0.09
0.09
0.01
0.0
0.14 O.ll0. I8
0.22 O.lS0.24
0.28 0.25% 0.33
0.30 0.280.33
0.30 0.280.31
0.38 0.320.43
0.38 0.3% 0.40
0.36 0.340.39
0.35 0.330.38
0.37 0.330.40
0.29 0.260.34
0.3 I 0.300.32
7
7
6
5
6
23 2223
30 2832
33 3134
39 3542
33 3234
7
13 1017
21 2023
but results
at 6 and Cl weeks after return
32 3034
to untreated
diet are the mean of two values.
3
6
8
0.22 -
0.19 -
TOXICOLOGY
OF CHLORINATED
PARAFFINS
525
found in rat abdominal fat (where the toxicity for the chlorinated paraffins and greatest tissue concentrations occurred) at that. they are unlikely to be mutagenic or concentrations which were not greater than carcinogenic. those in the diet, is of value. Campbell and McConnell (1980) found comparable conACKNOWLEDGMENTS centrations of chlorinated paraffins in human adipose tissue and certain foodstuffs, The authors thank Dr. T. F. McElligott, Mrs. S. E. providing direct evidence that at least in Moses, Dr. J. Styles, Mr. H. Bratt, Mr. G. H. Walker, Mr. M. J. L. Clapp, Dr. R. Gamer, Dr. D. A. Kinch, this respect, rats and man are comparable. Mr. M. Watson, and Mr. R. B. Wright for their With the limited data presented in Campbell’s paper, it is difficult to estimate the contributions to the work described in this paper. chlorinated partin concentration in total diet; a realistic upper estimate is 0.1 ppm REFERENCES which provides a several thousandfold AMES, B. N., MCCANN, J., AND YAMASAKI, E. (1975). safety factor between rats and man. AsMethods for detecting carcinogens and mutagens suming that man will respond in a similar with the Salmonella/mammalian-microsome mutaway to that seen in rats and dogs to the genicity test. Mural. Res. 31, 347-364. effects of chlorinated paraffins, and that CAMPBELL, I., AND MCCONNELL, G. (1980). Chloriman is more sensitive than these species nated paraffins in the environment. Environ. Sci. Technol., in press. to such effects, the safety margin is still CURLEY, A., BURSE, V. W., GRIM, M. E., JENNINGS, sufficiently large to enable one to conclude R. W., AND LINDER, R. E. (1971). Polychlorinated that the use of these chemicals presents little biphenyls: Distribution and storage in body fluids or no risk to man. and tissues of Sherman rats. Environ. Res. 4, Two important findings in the tissue reten481-495. tion studies in rats were that equilibrium DANIEL, J. W., AND GAGE, J. C. (1965). The absorption and excretion of butylated hydroxytoluene concentrations of radioactivity in abdominal (BHT) in the rat. Food Cosmet. Toxicol. 3,405-415. fat were similar to but never exceeded DOBBS, H. E. (1963). Oxygen flask method for the the dietary concentration of chlorinated assay of tritium, carbon-14 and sulfur-35 labelled paraffin, and that the levels of radioactivity compounds. Anal. Chem. 35, 783-786. in this tissue declined fairly rapidly after DRAIZE, J. H. (1959). Appraisal of the Safety of Chemicals in Food, Drugs and Cosmetics, pp. treatment stopped. These observations 46-59. Association of Food and Drug Officials of were made after feeding a diet containing a the United States, Washington, D.C. similar concentration of chlorinated paraf- KAY, J. H., AND CALANDRA, J. C. (1%2). Interpretafin to that estimated for man. Other organotion of eye irritation tests. J. Sot. Cosmet. Chem. chlorine compounds such as DDT (Laug 13, 281-289. LAUG, E. P., NELSON, A. A., FITZBURGH, 0. G., AND et al., 1950), Aldrin and Dieldrin (Quaife KUNZE, F. M. (1950). Liver cell alteration and DDT et al., 1967), and Aroclor 1254, a polystorage in the fat of the rat induced by dietary levels chlorinated biphenyl (Curley et al., 1971), of 1 to 50ppm DDT. J. Pharmacol. Exp. Ther. 98, have been shown to accumulate to con268-273. centrations in adipose tissue which are PURCHASE, I. F. H., LONGSTAFF, E., ASHBY, J., STYLES, J. A., ANDERSON, D., LEFEVRE, P. A., several times greater than the dietary conAND WESTWOOD, F. R. (1976). Evaluation of six centration fed to animals. This is an imshort term tests for detecting organic chemical portant difference between chlorinated carcinogens and recommendations for their use. paraffins and the other substances, and is an Nature (London) 264, 624-627. influential factor in the consideration that QUAIFE, M. L., WINBUSH, J. S., AND FITZHUGH, 0. G. (1967). Survey of quantitative relationships between chlorinated paraffins present a low potential ingestion and storage of aldrin and dieldrin in risk to man. The other factors supporting animals and man. Food Cosmet. Toxicol. 5, 39-50. this conclusion, which have been estab- STYLES, J. A. (1977). A method for detecting carlished from the investigations reported in cinogenic organic chemicals using mammalian cells in culture. Brit. J. Cancer 36, 558-563. this paper, are the low acute and subacute